Anti β actin

Antibodies were purchased as follows: anti-NeuN (Millipore, MA, USA), anti-BrdU (Abcam, Cambridge, UK), anti-neuritin (R&D systems, MI, USA), anti- beta-actin (anti-β- actin, Santa Cruz, CA, USA), anti-synaptophysin (Millipore, MA, USA), anti-glial fibrillary acidic protein (anti-GFAP, Dakocytomation, Glostrup, Denmark) antibodies. Neuritin peptide was purchased from Abcam.

Leelamine (LEE, Figure 1A) was purchased from Cayman Chemical (Ann Arbor, MI, USA). LEE stock solution (10 mM) was prepared in EtOH, storage at −20 °C and finally diluted in cell culture medium for use. Anti-CXCR7 and anti-CXCR4 antibodies were purchased from abcam (Cambridge, UK). Anti-MnSOD, anti-fibronectin, anti-vimentin, anti-MMP-9, anti-MMP-2, anti-N-cadherin, anti-E-cadherin, anti-twist, anti-snail, anti-occludin, and anti-b-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-phospho-STAT3(Tyr705), anti-STAT3, anti-phospho-JAK1(Tyr1022/1023), anti-JAK1, anti-phospho-JAK2(Tyr1007/1008), and anti-JAK2 were purchased from Cell Signaling Technology (Beverly, MA, USA). Alexa Fluor^® 488 donkey anti-rabbit IgG (H+L) antibody and Alexa Fluor^® 594 donkey anti-mouse IgG (H+L) antibody was obtained from Life Technologies (Grand Island, NY, USA). The GSH/GSSG-Glo™ Assay kit was purchased from Promega (Madison, WI, USA).

Cell lysates were obtained using RIPA lysis buffer (50 mM Tris–Cl pH = 8.0, 150 mM NaCl, 1% Igepal Ca 630, 0.5% Sodium Deoxycholate, 1.0% SDS, 0,4% protein inhibitor cocktail, 1 mM sodium orthovanadate, 10 mM NaF, 10 mM B-mercaptoethanol). Protein concentration was measured with a Bio-Rad protein assay on a Synergy H4 plate reader. Samples were loaded to 4–20% SDS pre-casted gels (Bio-Rad TGX gels) and transferred to a nitrocellulose membrane (Bio-Rad). The membranes were blocked with 5% skimmed milk and incubated with primary (O/N at 4  °C) and secondary (1 h at room temperature) and visualised using SuperSignal (Perkin Elmer) on a ChemiDoc MP imaging system (Bio-Rad) and quantified using ImageLab 6.0 (Bio-Rad). Antibodies used were: anti-MnSOD2 (1:1.000 Enzo), anti-B-actin (1:10.000 Santa Cruz), Goat-anti-rabbit-peroxidase (GARPO, 1:000 Dako). Full membranes are shown in Additional file 1: Figure S1.

Anti-ErbB3 antibody RTJ2 (Abcam); anti-erbB-3/HER-3 antibody, clone 2F12 (Millipore); mouse anti-human c-erbB-3 (BD Biosciences); phospho-HER3/ErbB3 (Tyr1289) (21D3) rabbit mAb, anti-b-actin, anti-b-tubulin (Santa Cruz); anti-GAPDH, anti-AKT, anti-pAKT, anti-pERK1/2, anti-p44/42 MAP kinase (Cell Signaling Technology) were used in the study. Secondary antibodies were purchased from Jackson ImmunoResearch or Invitrogen. DNaseI-RNase free and Ribonucleoside-vanadyl complex were from NEB. Human tumor cell lines used (BT-549, BT-474, Hs587T, MCF-7, MCF10A, SKBR-3, MDA-MB-231, MDA-MB-468 and HEK293) were from ATCC and grown in RPMI or D-MEM containing 10% FBS. The HER3 neutralizing antibodes (HER3Mabs) were produced in our laboratory and described previously [20 (link), 36 (link)]. Breefly, the HER3 neutralizing antibodies HER3Mabs are a monoclonal antibodies with backbone of IgG1. HER3Mabs were expressed in HEK293 freestyle cells (Life Technologies) and purified to above 95% purity using protein A/G affinity chromatography. Antibody purity was verified by protein gel electrophoresis and antibody binding was confirmed by ELISA binding assays, flow cytometry analysis. The ability to inhibit HER3 phosphorylation upon NRG-1 activation were verified by ELISA and WBs as described by our group previously [19 (link)].

Whole-cell extracts were obtained from cell pellets lysed in 1x Laemmli Sample Buffer (BIO-RAD) supplemented with 2-mercaptoethanol. Proteins were resolved on SDS-PAGE and transferred to nitrocellulose membranes (BIO-RAD). Membranes were blocked 1 hour in 5% Blotting-Grade Blocker (BIO-RAD) in 1× TBST) and incubated at 4°C overnight with anti-cytokeratin 10 (Santa Cruz Biotechnology; sc-52318), anti-E7 (Santa Cruz Biotechnology, sc-6981) or anti-b-actin (Santa Cruz Biotechnology; sc-47778) primary antibodies. After incubation, membranes were washed 3 × 15 minutes in 1× TBST wash buffer. Membranes were then incubated with horseradish peroxidase–tagged goat anti-mouse and goat anti-rabbit secondary antibodies (1:2500, Jackson ImmunoResearch) at room temperature for 1 hour, washed 3 × 15 minutes in 1× TBST. Signals were detected by enhanced chemiluminescence (Thermo Scientific). Equal protein loading was confirmed by probing with β-actin monoclonal antibody.